Calcium channels are membrane-spanning, multi-subunit proteins that allow controlled entry of Ca.sup.2+ ions into cells from the extracellular fluid. Cells throughout the animal kingdom, and at least some bacterial, fungal and plant cells, possess one or more types of calcium channel.
The most common type of calcium channel is voltage dependent. In a voltage-dependent channel, the "opening" to allow an influx of Ca.sup.2+ ions into the cells to begin, requires a depolarization to a certain level of the potential difference between the inside of the cell bearing the channel and the extracellular medium bathing the cell. The rate of influx of Ca.sup.2+ into the cell depends on this potential difference. All "excitable" cells in animals, such as neurons of the central nervous system, peripheral nerve cells, and muscle cells, including those of skeletal muscles, cardiac muscles, and venous and arterial smooth muscles, have voltage-dependent calcium channels.
Calcium channels are physiologically important because the channels have a central role in regulating intracellular Ca.sup.2+ levels. These levels are important for cell viability and function. Thus, intracellular Ca.sup.2+ concentrations are implicated in a number of vital processes in animals, such as neurotransmitter release, muscle contraction, pacemaker activity, and secretion of hormones and other substances.
The rabbit skeletal muscle calcium channel is the most well-characterized of the calcium channels identified to date. Biochemical analysis of the calcium channel purified from rabbit skeletal muscle revealed that it consists of two large subunits, of between about 130 and about 200 kilodaltons ("kD") in molecular weight, and a number (generally thought to be one to three) of different smaller subunits, of less than about 60 kD in molecular weight. At least one of the larger subunits and possibly some of the smaller are glycosylated. Some of the subunits are capable of being phosphorylated.
The two large subunits of voltage-dependent calcium channels are designated herein the ".alpha..sub.1 -subunit" and the ".alpha..sub.2 -subunit".
The rabbit skeletal muscle calcium channel .alpha..sub.1 -subunit is not detectably changed in molecular weight when treated with dithiothreitol ("DTT") or with enzymes which catalyze removal of N-linked sugar groups from glycosylated proteins. The .alpha..sub.1 -subunit has a molecular weight of about 150 to about 170 kD when analyzed by sodium dodecylsulfate ("SDS")-polyacrylamide gel electrophoresis ("PAGE") after isolation from mammalian muscle tissue and has specific binding sites for various 1,4-dihydropyridines ("DHPs") and phenylalkylamines.
The molecular weight of the .alpha..sub.2 -subunit of the rabbit skeletal muscle calcium channel is at least about 130-150 kD, as determined by SDS-PAGE analysis in the presence of DTT after isolation from muscle tissue. However, in SDS-PAGE under non-reducing conditions (in the presence of N-ethylmaleimide), the .alpha..sub.2 -subunit migrates with a band of about 160-190 kD. The smaller fragments (of about 30 kD), which appear to be released upon reduction, are derived from the primary translation product of the .alpha..sub.2 subunit transcript. There is evidence that the .alpha..sub.2 -subunit and the corresponding fragment produced under reducing conditions are glycosylated with at least N-linked sugars and do not have specified binding sites for 1,4-dihydropyridines and phenylalkylamines that are known to bind to the .alpha..sub.1 -subunit.
The .beta.-subunit of the rabbit skeletal muscle calcium channel has recently been characterized as having an apparent molecular mass of 52-65 kD (as determined by SDS-PAGE analysis). It is comprised of consensus phosphorylation sites and has been shown by biochemical methods to be phosphorylated. This subunit is insensitive to reducing conditions.
The .gamma.-subunit of the calcium channel has not been observed in all purified preparations, depending on the source of material analyzed, the investigating laboratory, and so on. The native material appears to be a glycoprotein with an apparent molecular mass of 30-33 kD, as determined by SDS-PAGE analysis. The native protein is believed to be glycosylated since its apparent molecular mass decreases after digestion with neuraminidase followed by endoglycosidase F.
Multiple types of calcium channels have been detected based on electrophysiological and pharmacological studies of various mammalian cells from various tissues (e.g., skeletal muscle, cardiac muscle, lung, smooth muscle and brain) [Bean, B. P., Annu. Rev. Physiol. 51:367-384 (1989) and Hess, P., Annu. Rev. Neurosci. 56:337 (1990)]. These different types of calcium channels have been broadly categorized into four classes, L-, T-, N-, and P-type, distinguished by current kinetics, holding potential sensitivity and sensitivity to calcium channel agonists and antagonists. Four subtypes of neuronal voltage-dependent calcium channels have been proposed [Swandulla, D. et al., Trends Neurosci 14:46 (1991)].
Characterization of a particular type of calcium channel by analysis of whole cells is severely restricted by the presence of mixed populations of different types of calcium channels in the majority of cells. This hindrance is also a drawback in attempting to discern whether a calcium current with properties that preclude categorization on the basis of these four broad classes is generated by a new type or subtype of calcium channel or a previously classified channel that is obscured by contaminating currents. Although single-channel recording methods can be used to examine individual calcium channels, such analysis reveals nothing about the molecular structure or biochemical composition of the channel. Furthermore, in this type of analysis, the channel is isolated from other cellular constituents that might be important for natural functions and pharmacological interactions.
Structural features of calcium channels can also be used in evaluation and characterization of different types of calcium channels. However, large amounts of pure channel protein are required to understand, at the molecular level, the nature of the subunits and their various interactions, for example, with one another, with the cell membranes across which the channels allow Ca.sup.2+ ions to pass, with Ca.sup.2+ and other ions, and with low molecular weight compounds such as drugs (pharmacological agents) that affect channel function. Due to the complex nature of these multi-subunit proteins, the varying levels of calcium channels in tissue sources of the protein, the presence of mixed populations of calcium channels in tissues, and the modifications of the native protein that can occur during the isolation procedure, it is extremely difficult to obtain large amounts of highly purified, completely intact calcium channel protein.
Characterization of the gene or genes encoding calcium channels provides another means of characterization of different types of calcium channels. The amino acid sequence determined based on the complete nucleotide sequence of the coding region of a gene encoding a calcium channel protein represents the actual primary structure of the protein. Furthermore, secondary structure of the calcium channel protein and the relationship of the protein to the membrane may be predicted based on analysis of the primary structure. For instance, hydropathy plots of the .alpha.1 subunit protein of the rabbit skeletal muscle calcium channel indicate that it contains four internal repeats, each containing six putative transmembrane regions. [Tanabe, T. et al., Nature 328:313 (1987).]
The cDNA and corresponding amino acid sequences of the .alpha.1, .alpha.2, .beta. and .gamma. subunits of the rabbit skeletal muscle have been determined [see Tanabe et al., Nature 328:313-318 (1987), Ellis et al., PCT Publication No. WO 89/09834, Ruth et al., Science 245:1115-1118 (1989), and allowed U.S. patent application Ser. No. 482,384, filed Feb. 20, 1990, (the disclosure of which is hereby incorporated by reference), respectively]. In addition, the cDNA and corresponding amino acid sequences of .alpha.1 subunits of rabbit cardiac muscle [Mikami, A. et al., Nature 340:230-233 (1989)] and lung [Biel, M., FEBS Letters 269:409-412 (1990)] calcium channels have been determined. Recently, a rabbit brain calcium channel (designated the BI channel) cDNA was isolated [Mori, Y. et al., Nature 350:398-402 (1991)]. The amino acid sequences deduced from the rabbit skeletal muscle, rabbit cardiac muscle, and rabbit lung cDNAs and the rabbit brain BI cDNA indicate that these proteins share some general structural features. However, the sequences share, at most, .sup..about. 60% homology and appear to be encoded by a minimum of three distinct genes. These findings correlate with the varied intensities of hybridization of the rabbit skeletal muscle calcium channel .alpha.1 subunit cDNA to rabbit genomic DNA fragments as reported by Ellis et al., Science 241:1661-1664 (1988).
Interestingly, partial cDNAs encoding portions of several different subtypes of the calcium channel .alpha.1 subunit have been isolated from rat brain [Snutch, T. et al., Proc. Natl. Acad. Sci U.S.A. 87:3391-3395 (1990)]. These are referred to as rat brain class A, B, C and D cDNAs. More recently full-length rat brain class A [Starr, T. et al., Proc. Natl. Acad. Sci. U.S.A. 88:5621-5625 (1991)] and class C [Snutch, T. et al., Neuron 1:45-57 (1991)] cDNAs have been reported. Although the amino acid sequence encoded by the rat brain class C cDNA is approximately 95% identical to that encoded by the rabbit cardiac muscle calcium channel .alpha.1 subunit cDNA, the amino acid sequence encoded by the rat brain class A cDNA shares only 33% sequence identity with the amino acid sequences encoded by the rabbit skeletal or cardiac muscle .alpha.1 subunit cDNAs. A cDNA encoding another calcium channel .alpha.1 subunit was also recently reported [Hui, A. et al., Neuron 7:35-44 (1991)]. The amino acid sequence encoded by this cDNA is .sup..about. 70% homologous to the proteins encoded by the rabbit skeletal and cardiac muscle calcium channel cDNAs.
A cDNA closely related to the rat brain class C .alpha.1 subunit cDNA and partial cDNA sequences closely related to other cDNAs encoding apparently different calcium channel .alpha.1 subunits have also been described [see Snutch, T. et al., Neuron 7:45-57 (1991), Perez-Reyes, E., Wei, X., Castellano, A. and Birnbaumer, L., J. Biol. Chem. 365:20430 (1990), and Hui, A. et al, Neuron 7:35-44 (1991)]. Evidence suggests that the closely related cDNA sequences, which are identical to some of the previously isolated .alpha.1 subunit cDNAs except in certain limited areas, represent variants generated by alternative splicing of a primary gene transcript.
Although the existence of numerous types and subtypes of calcium channel .alpha.1 subunits with a broad range of homologies is of interest, this information may be of limited utility in the absence of the knowledge of the functional characteristics of the calcium channels containing these different .alpha.1 subunits. Insufficient information is available to predict or discern, based on the primary structure of the .alpha..sub.1 subunits, the functional or pharmacological properties of voltage-dependent calcium channels containing the different .alpha..sub.1 subunits. Therefore, attempts to recombinantly express mammalian calcium channel .alpha.1 subunits have been reported.
To date, successful recombinant expression has been reported for only three of the six or seven different rabbit or rat .alpha..sub.1 subunit cDNAs referred to in the preceding paragraphs. Perez-Reyes et al., Nature 340:233-236 (1989) have described the presence of voltage-dependent calcium currents in murine L cells transfected with the rabbit skeletal muscle calcium channel .alpha.1 subunit cDNA. These currents were enhanced in the presence of Bay K8644 (a known calcium channel agonist). Bay K8644-sensitive Ba.sup.2+ currents have been detected in oocytes injected with in vitro transcripts of the rabbit cardiac muscle calcium channel .alpha.1 subunit cDNA [Mikami, A. et al., Nature 340:230-233 (1989)]. These currents were substantially reduced in the presence of the calcium channel antagonist nifedipine. Significantly, the barium currents of an oocyte co-injected with transcripts of the rabbit cardiac muscle calcium channel .alpha.1 subunit cDNA and the rabbit skeletal muscle calcium channel .alpha.2 subunit cDNA were more than 2-fold larger than those of oocytes injected with transcripts of the rabbit cardiac calcium channel .alpha. 1 subunit cDNA. Similar results were obtained when oocytes were co-injected with transcripts of the rabbit lung calcium channel .alpha.1 subunit cDNA and the rabbit skeletal muscle calcium channel .alpha.2 subunit cDNA, i.e., the barium current was enhanced relative to that detected in oocytes injected with transcripts of the rabbit lung calcium channel .alpha.1 subunit cDNA only [Biel, M. et al, FEBS Letters 269:409-412 (1990)]. Most recently, Mori et al., Nature 350:398-402 (1991) report the presence of inward barium currents in oocytes injected with in vitro transcripts of the rabbit brain BI channel cDNA. These currents were increased by two orders of magnitude when in vitro transcripts of the rabbit skeletal muscle calcium channel .alpha.2-, .beta.-, or .alpha.2-, .beta.- and .gamma.-subunits were co-injected with transcripts of the BI cDNA. Barium currents in oocytes co-injected with transcripts of the BI cDNA and the rabbit skeletal muscle calcium channel .alpha.2 and .beta. cDNAs were unaffected by the calcium channel antagonists nifedipine or .omega.-CgTx and inhibited by Bay K8644 and crude venom from Agelenopsis aperta.
The results of studies of recombinant expression of rabbit calcium channel .alpha.1 subunit cDNAs and transcripts of the cDNAs indicate that the .alpha.1 subunit forms the pore through which calcium enters cells. However, the relevance of the barium currents generated in these recombinant cells to the actual current generated by calcium channels containing as one component the respective .alpha.1 subunits in vivo is unclear. Because addition of in vitro transcripts of rabbit skeletal muscle calcium channel .alpha.2 and/or .beta. and .gamma. cDNAs significantly enhanced the barium currents in the recombinant cells, it appears that to completely and accurately characterize and evaluate different calcium channel types, it is essential to examine the functional properties of recombinant channels consisting of all the subunits as found in vivo. However, cDNAs encoding .alpha.2-, .beta.- and .gamma.-subunits from any of the rabbit or rat tissues besides rabbit skeletal muscle tissue are not available for use in such studies. The usefulness of rabbit skeletal muscle calcium channel .alpha.2- and .beta.-subunit cDNAs in attempting to recombinantly evaluate different calcium channel types is extremely limited. Although others have suggested that the .beta.- and .alpha.2-subunits of rabbit calcium channels from different tissues are essentially identical [Mori, Y. et al., Nature 350:398 (1991)], as described herein, different forms of .alpha.2-and .beta.-subunits, arising from alternative splicing of the corresponding genes, are expressed in human brain, skeletal muscle and aorta. Therefore, in evaluating specific calcium channel types by examination of recombinantly expressed channels, it is most valuable to express cDNAs encoding calcium channel subunits from the same type of tissue.
It appears that calcium channels, specifically human calcium channels, can be relevant in certain disease states. A number of compounds useful in treating various cardiovascular diseases in animals, including humans, are thought to exert their beneficial effects by modulating functions of voltage-dependent calcium channels present in cardiac and/or vascular smooth muscle. Many of these compounds bind to calcium channels and block, or reduce the rate of, influx of Ca.sup.2+ into the cells in response to depolarization of the cell membrane.
An understanding of the pharmacology of compounds that interact with calcium channels in other organ systems, such as the central nervous system ("CNS"), and the ability to rationally design compounds that will interact with these specific subtypes of human calcium channels to have desired therapeutic, e.g., treatment of neurodegenerative disorders, effects have been hampered by an inability to independently determine how many different types of calcium channels exist or the molecular nature of individual subtypes, particularly in the CNS, and the unavailability of pure preparations of specific channel subtypes, i.e., systems to evaluate the specificity of calcium channel-effecting compounds.